TyrZ Oxidation at Liquid Helium Temperatures

As previously mentioned, NIR illumination on PSII samples poised in S2was expected to trans- fer of an electron hole from the WOC to TyrZ, thus resulting in a S2TyrZ/S1TyrZ◦ difference spectrum. PSII samples were poised in the S2 state via illumination at 200 K for spinach PSII membranes andT.vulcanuscores, and 235 K for spinach PSII core; we chose to illuminate our spinach PSII cores at 235 K as we have previously observed that 200 K resulted in carotenoid oxidation in those samples, implying that S2formation was less efficient under those conditions. The resulting difference signals are shown in Figure 3.8.

FIGURE3.8: S2QA–/S1QAdifference signals from green illumination at 200 K and 235 K, onT.vulcanus

PSII core samples, spinach PSII membrane pellet samples and spinach PSII core samples. The spinach PSII core signals are weaker because those samples were relatively dilute.

However, it is demonstrated that no NIR-induced signals could be detected in PSII core samples poised in S2, other than those attributable to the ZnSe windows. The 10 K NIR illumination dif- ference spectra obtained from PSII cores are shown in Figure 3.9, and the double-difference with the ZnSe signal revealed no underlying features that can be attributed to PSII. The equivalent NIR-induced difference signal from PSII membranes is shown in Figure 3.3, but was excluded here since that signal contains strong contributions from P700+/P700.

FIGURE 3.9: FTIR difference signals from NIR illumination at 10 K, on PSII core samples poised in S2. (A) NIR-induced signal from spinach PSII cores, (B) NIR-induced signal fromT.vulcanuscores, (C)

spinach PSII cores signal minus ZnSe signal, (D)T.vulcanuscores signal minus ZnSe signal. The features observed in theT.vulcanussignal are water bands that arise due to small amounts of water condensation

In Figure 3.10, it is shown that green illumination on dark-adapted samples at 10 K induced a transient PSII signal that decayed over time; T.vulcanusPSII core samples produced a signifi- cantly stronger FTIR difference signal than spinach PSII membrane samples, but this signal has largely the same difference features. The signal could be repeatedly re-induced by illumination, and may correspond to the FTIR signature of the EPR S1TyrZ◦split signal. A positive feature at ∼1480 cm−1 characteristic of Q_Areduction was also observed. An equivalent difference signal from spinach PSII cores is not shown here, due to the relatively weak difference signal that had been obtained from those samples thus far.

FIGURE 3.10: Transient signals from PSII induced by 10 K continuous green illumination, in spinach PSII membranes andT.vulcanusPSII cores (A). The decay of the transient signal in aT.vulcanussample is shown in (B). To avoid contributions from the ZnSe signal, only the region above 1400 cm−1was ex- amined. These signals were also corrected for the illumination-induced baseline shift using the Concave Rubberband baseline correction method. In (A), theT.vulcanusPSII core signal was averaged over six

The transient signal fromT.vulcanussamples and the signal generated from sampleCin the pre- vious section are compared in Figure 3.11. The differential feature at 1756(+)/1749(-) cm−1 is much more prominent in the transient signal than the nominal “Q_A–/Q_A” signal fromC. Differ- ences in the intensity of the 1719(+) cm−1, 1700(+) cm−1, 1660(+) cm−1, 1649 cm−1, 1556(+) cm−1 and 1541(+) cm−1 features were also observed, amongst other more subtle differences. However, note that the baseline slope caused by ZnSe photosensitivity may not be completely removed by the rubberband baseline correction method in theT.vulcanussignal, which can af- fect the apparent intensity of the difference signal features.

While a double-difference between these signals would, in theory, reveal the difference features of the transient donor, distortions due to subtle inter-organism differences are likely to interfere with the signal, and we do not have a 10 K Q_A–/Q_Adifference spectrum fromT.vulcanusdue to the limited amount ofT.vulcanussample available. The transient signal from spinach PSII membranes on the other hand, is comparatively weak and has low SNR; a double-difference using this signal would be dominated by noise.

FIGURE3.11: The transient signal fromT.vulcanuscompared to the “QA–/QA” signal observed in sample

C, which was treated with 20 mM Asc. The signal fromCwas scaled down so that the QA–/QAfeature

at∼1480 cm−1_{in both spectra were matched in amplitude.}

EPR experiments showed that a well-known split signal was indeed, induced in spinach PSII cores by the illumination protocols used in FTIR, as shown in Figure 3.12.20,22,24,25,35The de- crease in the S2 multiline signal following NIR illumination are in-line with observations made

by Petrouleas et al.22It is not immediately clear as to why a corresponding FTIR difference sig- nal was not observed. We do note however, that the NIR illumination in the EPR experiment was 30 minutes as opposed to 1 minute in the FTIR experiment, as it was necessary to compensate for the substantial increase in sample thickness.

FIGURE 3.12: The stable and transient split signals, measured in spinach core samples using EPR. The NIR illumination was performed over 30 minutes, and the signal induced by continuous green illumina- tion was allowed to decay for 10 minutes prior to the “after illumination” measurement. (A) The split signals in the 3090 – 3595 G region. (B) The split signal in the 2450 – 4250 G region, which shows that the amplitude of the S2multiline signal was decreased by NIR illumination. EPR parameters: Microwave